Fermentation is an important technology for the biocatalytic conversion of renewable feedstocks into desirable products. Carbohydrates are traditional feedstocks in the fermentation industry. It is often the case that carbohydrates used as a substrate contribute more to the cost of manufacture than any other single component. Depending on the particular process, from 25 to 70% of the total cost of fermentation may be due to the carbohydrate source. (Crueger and Crueger, Biotechnology: A Textbook of Industrial Microbiology, Sinauer Associates: Sunderland, Mass., pp 124-174 (1990); Atkinson and Mavituna, Biochemical Engineering and Biotechnology Handbook, 2nd ed.; Stockton Press: New York, pp 243-364 (1991)). For such economic reasons, highly purified glucose or sucrose can seldom be used as a substrate.
Starch, a carbohydrate, is a mixture of two different polysaccharides each consisting of chains of linked, repeating monosaccharide (glucose) units. The mixture consists of two separate polysaccharides, amylose and amylopectin. Amylose is a linear polysaccharide with glucose units connected exclusively through α(1,4) glycosidic linkages. Glucose units in amylopectin are also linked through α(1,4) glycosidic linkages, and additionally are linked through α(1,6) glycosidic linkages, about one every 30 glucose residues. The ratio of amylopectin to amylose in starch varies from one plant species to another, but is generally in the range of 3-4 to 1 (Kainuma, pp 125-150 in Starch; Whistler, Bemiller, and Pashcall eds., Academic Press, Orlando, Fla. (1984)).
Commercial starch is produced primarily through the wet milling process. The final products from a wet mill, however, include very little unprocessed starch. By far, the majority of products made are in the form of fully processed starch (monosaccharides, including glucose) or smaller degradation products derived from starch. Typically, an amylase enzyme is used to break starch into smaller chains (Blanchard, Technology of Corn Wet Milling (1992), Elseiver, Amsterdam, The Netherlands, pp. 174-215). Various commercial sources of α-amylase exist, but, regardless of enzyme source, reaction products are generally the same with respect to size and linkage-type. Amylase digestion of starch results in a product known as a limit dextrin that includes small starch chains containing 2-10 glucose units (oligosaccharides). Because amylase cannot hydrolyze the α(1,6) glycosidic linkages in amylopectin, limit dextrins contain both α(1,4)- and α(1,6)-linked glucose oligosaccharides. Alternatively, raw starch may be treated by non-enzymatic means (for example, by acid hydrolysis) to produce starch products substantially similar to limit dextrin.
In the wet milling industry, limit dextrins are further processed into glucose for use as a carbon source for fermentations to produce various chemicals, commercial enzymes, or antibiotics. Relatively pure glucose is preferred as a carbohydrate source when the popular biocatalyst, Escherichia coli, is used in the fermentation process. This is because E. coli does not utilize components of limit dextrins (i.e., panose, isomaltose, and high molecular weight oligosaccharides with chains larger than about ten α(1,4)-linked glucose units) that are commonly contained in alternate low-cost fermentation media (Lin, Escherichia coli and Salmonella typhimuium, pp. 245-265, Neidhardt, ed.; American Society for Microbiology, Washington, D.C. (1987)). Glucose oligomers containing α(1,6)-linkages are not transported into the cell and E. coli does not produce an enzyme that degrades this material when supplied extracellularly (Palmer et al., Eur. J. Biochem. 39:601-612 (1973)).
Making relatively pure glucose from starch that is suitable for use by E. coli requires many process steps and additional enzymes, adding significantly to the cost of product manufacture.
Thus, the problem to be solved is the lack of a process to utilize low-cost starch products in large-scale fermentative production processes. An ability to more completely ferment low cost, partially degraded starch would lower the cost of manufacture for products made through fermentation.